PDF U.S. Pharmacopeia National Formulary USP 39 NF 34

2016

U.S. Pharmacopeia National Formulary

USP 39 NF 34

USP 39 Published General Chapter Residual Host Cell Protein Measurement in Biopharmaceuticals

The official version can be found in the USP?NF. The USP?NF is subscription based publication. For more information on how to access the USP?NF click here.

Official: May 1, 2016

2016

USP 39 THE UNITED STATES PHARMACOPEIA

NF 34

Volume 1

THE NATIONAL FORMULARY

By authority of the United States Pharmacopeial Convention Prepared by the Council of Experts and its Expert Committees

Official from May 1, 2016

The designation on the cover of this publication, "USP NF 2016," is for ease of identification only. The publication contains two separate compendia: The United States Pharmacopeia, Thirty-Ninth Revision, and The National Formulary, Thirty-Fourth Edition.

THE UNITED STATES PHARMACOPEIAL CONVENTION 12601 Twinbrook Parkway, Rockville, MD 20852

1416 ?1130? Nucleic Acid-Based Techniques / General Information

USP 39

Although safety concerns regarding residual DNA impurities are not as prominent as they once were, the levels of residual DNA in any bioprocess remain a key quality attribute and help define the process.

General Chapters

?1132? Residual Host Cell Protein Measurement in Biopharmaceuticals

1. INTRODUCTION AND SCOPE

Many medicinal products are produced through recombinant technology via a host cell (e.g., bacteria, yeast, or mammalian, insect, or plant cell lines). During the manufacture of such products, some amount of non-product, host cell-derived material will inevitably be introduced into the process stream. This process results in a mixture of the desired product and host cellderived impurities, including host cell proteins (HCPs), and other process-related impurities that will be targeted for clearance through bioprocessing.

Residual HCPs have the potential to affect product quality, safety, and efficacy; therefore, the quantity of HCPs should be low. The product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible.

The primary concern with HCPs in biopharmaceutical products is their potential to induce anti-HCP antibodies that could induce a clinical effect in patients. In addition, HCPs may possibly act as adjuvants, which can induce anti-drug antibodies that can affect the safety or efficacy of the drug. A more extensive discussion of immunogenicity and its effect on preclinical and clinical studies is described in USP general chapter Immunogenicity Assays--Design and Validation of Immunoassays to Detect Anti-Drug Antibodies ?1106?. HCPs can also have a direct effect on the quality of the product itself. For example, proteolytic HCPs, even in minute quantities, can cleave the desired protein product over time, reducing or eliminating biological potency or altering stability.

This chapter focuses on HCP immunoassays for recombinant therapeutic products. It does not address products such as vaccines or gene-, cell-, or tissue-based therapies, although the general principles discussed may apply to the measurement of HCPs in these products. The design and validation of immunoassays for HCPs involve unique and significant challenges due to: 1) the wide variety of possible HCPs in medicinal products; 2) the general use of polyclonal antibody reagents to detect them; 3) the lack of exactly matched standards for quantitation; 4) in some cases, a considerable effect from sample dilution effects; and 5) inherent limitations to measure single HCP species.

The chapter includes assay development strategies throughout the product and process development lifecycle, and it describes approaches to demonstrate that the assay is fit for use (e.g., illustrates unit operation clearance of HCPs, lot release). Because of the complexity of HCP immunoassays, careful development and characterization of critical reagents are required, particularly for the immunogen used to elicit the anti-HCP antibodies, the antibody reagent(s), and the assay HCP standard. Because HCP testing is an essential part of process development and product quality control, HCP testing is also discussed in conjunction with regulatory requirements and other considerations for guidance on an overall control strategy for HCPs. A brief outline of the general chapter follows:

1. Introduction and Scope 1.1 Considerations for Manufacturing, Characterization, and Consistency

2. Terminology 3. HCP Immunoassay Methods

3.1 The Assay Development Cycle 3.2 Development and Characterization of HCP Reagents 3.3 Immunoassay Method Development and Qualifying as Fit for Use 4. HCP Immunoassay Method Validation 4.1 Accuracy 4.2 Sensitivity and Assay Range 4.3 Sample Linearity 4.4 Specificity 5. Supporting Technologies for Residual HCP Detection, Identification, and Measurement 5.1 Considerations for Electrophoretic Methods 5.2 Considerations for Western Blot Methods 5.3 Considerations for Chromatographic and Proteomic Methods 5.4 Concluding Remarks on Supporting Technologies for HCPs 6. Use of HCP Immunoassays for Process Development, Characterization, and Validation 6.1 Assays for Individual HCPs 6.2. Control Strategy 7. Summary and Conclusions 8. Bibliography

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General Information / ?1132? Residual Host Cell Protein 1417

General Chapters

1.1 Considerations for Manufacturing, Characterization, and Consistency

Different cell-based expression systems are used to manufacture medicinal products, such as bacteria (Escherichia coli, Pseudomonas fluorescens), yeast (Saccharomyces cerevisae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary (CHO), mouse myeloma cell line NSO, and others), insect cells (baculovirus-infected Spodoptera frugiperda cells), and plant cells (tobacco, Arabidopsis, rice). The particular HCP profile is unique and specific to the particular host cells under specific culture conditions and manufacturing processes. HCPs can vary in pI (~3?11) and hydrophobicity, and HCPs display a wide range of molecular weights (from ~5 kDa to at least ~250 kDa), depending on the host cell and manufacturing process used. The number of HCPs in upstream samples can run anywhere from several hundred to more than one thousand proteins, depending on the host cell and culture conditions. Although many cellular hosts have been used in biopharmaceutical manufacturing historically, the most experience has been gained using E. coli and the mammalian cells CHO, NSO, SP2/0, and human embryonic kidney cell line HEK293. The guidance in this chapter draws most heavily from the experience with these expression systems; however, general principles apply broadly to any host cell system.

In mammalian cells, the recombinant protein is typically secreted from the cells into the cell culture fluid (CCF), along with many of the HCPs. However, it has been observed that intracellular protein trafficking may not proceed in a normal fashion in production cultures. For example, proteins usually associated with intracellular organelles, such as lysosomes, may be found in the CCF of largely viable cell cultures, because the clones have been selected for maximum protein export. In addition, as some of the cells die, their soluble, intracellular proteins are released into the CCF. Some harvest operations also lyse cells; therefore, the resulting harvested CCF typically contains both secreted and intracellular HCPs. While this mixture of proteins incubates in the fermenter, additional changes in the HCP population may occur, for example, as the result of enzymatic activity (e.g., proteinases or sialidases).

HCP assays provide important information about the composition of the material entering the downstream recovery process and how each purification step affects HCP clearance. In some cases, HCPs can even bind to, and co-purify with, certain products. Process characterization and validation studies are needed to show which process steps remove HCPs and also to demonstrate the robustness of these steps for consistently removing HCPs. As such, HCP assays are an essential part of purification process development and help ensure manufacturing consistency. Lastly, reproducible and reliable HCP assays may be required to measure residual HCPs remaining in the drug substance (DS) used to make drug product (DP) that is delivered to the patient. HCP levels should be measured in: 1) preclinical lots used in toxicology assessment, 2) all lots during clinical development, and 3) process validation samples from the final manufacturing process. After approval, HCP monitoring may be required as an element of the control system. Subsequent sections of this chapter discuss in more detail the use of HCP assays in process validation and in a good manufacturing practices (GMP) control system.

2. TERMINOLOGY

To help establish a common nomenclature in the literature and with regulatory agencies, Table 1 lists common terms with their definitions (indicating how they are used in this chapter) in addition to synonyms that have been used historically. Note that the term "platform" indicates that the same set of standards and reagents is used within a company to test a variety of products made from the same type of expression system (e.g., CHO cells) grown under similar upstream conditions. In the case of platform HCP assays, the antibodies to HCP are obtained from animals immunized with HCP antigens generated from a common upstream process that is applicable to many products, even if the downstream purifications are different. This approach allows the knowledge from prior products to be leveraged. Justification that an assay is suitable for a new product, using the same expression system and common upstream conditions, is therefore often relatively straightforward.

The HCP immunogen used to generate platform anti-HCP antibodies and used often as the assay calibration standard is, by design, comprised of a broad set of HCPs. In contrast, the qualifier "process-specific" indicates that the immunogen/standard has been prepared from a set of HCPs unique to a given process (either a unique upstream cell culture process or a unique downstream purification process). Process-specific assays are, therefore, limited in their utility, and each must be fully qualified for each process. Process-specific immunogens and calibration standards are, by intent, more narrow and specific to a given process. "Commercially available" assays produced by vendors are often derived from a combination of strains and harvest/ purification procedures, and these assays are intended to have a broad application; but these commercially available assays are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency.

Table 1. HCP-associated Terminology

Term

Definition

Historical Synonyms

Commercial- Available to the public for commercial sale; typically a combination of upstream isolates Generic ly available and corresponding antibodies made by the vendor and sold as reagents or kits.

a In some cases, both purifications are performed, typically the protein A/G first, then the HCP affinity. b Although ppm has been used historically, it is not advised because this term is used to reflect mass per unit volume for other types of tests. It is recognized that ng is used conventionally as a value derived from interpolation from an HCP standard curve (in units of ng/mL), where the signal is reflective of antibody binding and, unlike the therapeutic protein concentration measurement, does not strictly reflect the mass of HCP that may be present.

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General Chapters

Table 1. HCP-associated Terminology (Continued)

Term

Definition

Historical Synonyms

Platform

The same set of an HCP standard and antibodies is developed with a company's propriet- Custom, in-house, proprietary ary host cell strain and used broadly within a type (e.g., CHO) across several products when the upstream conditions are similar.

Upstream process specific

An assay designed from material where the upstream culture process deviates significantly from the platform. This is generally before any purification and may be applied to more than one product if these parameters are similar.

Custom, in-house, product-specific, proprietary

Downstream process specific

An assay designed from materials where the downstream unit operations are used to enrich the HCP population. This may be applied to more than one product if these parameters are similar. This is rarely used today and is not recommended except for certain products with exceptional downstream processing.

Custom, in-house, product-specific, proprietary

Assay for an individual HCP

An assay using a standard composed of an identified, single, known HCP and its specific antibody/antibodies.

Single analyte assay or Custom, HCP-specific

Coverage

Describes the assessment of how completely a population of polyclonal antibodies recognize the population of HCPs. The coverage assessment may be made on the HCP population used as the HCP antigen or from the product production culture.

Qualification Demonstration of suitability of analytical methods (including reagents used in these methods) for their intended application to a given process and in-process samples.

Protein A/G- Affinity purification of antibodies with immobilized Protein A or Ga affinity purification

Affinity chromatography

HCP-affinity Affinity purification of antibodies using immobilized HCP (antigen)a purification

Affinity chromatography, immunoaffinity chromatography

Null cell

The cell strain used for production that does not contain the product-specific genetic ele- Parental, blank, or mock-transfected cell ments; includes untransfected parental cells and cells transfected with the expression vector but without the product gene.

ng/mg

The numerical quantity (ratio) of HCP per product, where ng represents HCP mass and mg represents the product mass. It is calculated by dividing the HCP concentration (ng/mL) by the product protein concentration (mg/mL).

ppmb

a In some cases, both purifications are performed, typically the protein A/G first, then the HCP affinity. b Although ppm has been used historically, it is not advised because this term is used to reflect mass per unit volume for other types of tests. It is recognized that ng is used conventionally as a value derived from interpolation from an HCP standard curve (in units of ng/mL), where the signal is reflective of antibody binding and, unlike the therapeutic protein concentration measurement, does not strictly reflect the mass of HCP that may be present.

3. HCP IMMUNOASSAY METHODS

Immunoassay methods rely on antibodies that recognize, as broadly as possible, the population of HCPs entering the downstream purification process; therefore, the sandwich immunoassay, designed with polyclonal antibodies, is the workhorse of HCP monitoring and quantitation. This assay format offers a combination of high sensitivity, specificity, throughput, automation potential, rapid turnaround, quantitative results, and low cost per assay that is unmatched by any other currently available assay technology. Other immunoassay formats (e.g., competitive immunoassays) may or may not be suitable, because they lack either the specificity or the sensitivity afforded by the sandwich format. Although these methods result in a single HCP value for a given lot, the number can give greater weight to HCPs for which high-affinity antibodies are present in the reagent(s)--and no or low weight to HCPs which are either not recognized or recognized by low-affinity antibodies in the assay. For these reasons, orthogonal measures of product purity are often needed. More details on these methods can be found in 5. Supporting Technologies for Residual HCP Detection, Identification, and Measurement.

The basic principles and design of immunoassays are discussed in USP chapter Immunological Test Methods--Enzyme-Linked Immunosorbent Assay (ELISA) ?1103?. The format that is most commonly used for HCP testing is the sandwich immunoassay with detection systems such as colorimetric, electrochemiluminescent (ECL), chemiluminescent, radioactive, or others. Homogeneous immunoassays, including competitive assays, where all of the reagents are combined at once and the binding occurs in a single step without washing, may be problematic due to antigen excess leading to antibody insufficiency issues (discussed later in the chapter); therefore, these formats should be used with caution. The heterogeneous sandwich immunoassay format described in chapter ?1103? is generally preferred, because the dynamic range and sensitivity may be reduced in the homogeneous format. The formats, with their advantages and disadvantages, are discussed further in chapter Immunological Test Methods--General Considerations ?1102?. Data analysis is typically performed with a nonlinear fit of the sigmoidal curve generated by a wide range of standard concentrations, although some analyses may focus on the low end of the curve for greater sensitivity.

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